Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol

The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal generated by modified proteins. We also demonstrated a specific fluorescent emission of 2,2,2-trichloroethanol-labeled protein at 450 nm, with a 310 nm excitation, resulting from modification of both tryptophan and tyrosine residues. Following optimization, this protein quantification assay displayed superior sensitivity when compared to UV absorbance at 280 nm (A280), and enabled quantification beyond the linear range permitted by the Bradford method. This 100 μL assay displayed a sensitivity of 10.5 μg in a range up to at least 200 μg. Furthermore, we extended the utility of this method through the development of a 20 μL low-volume assay, with a sensitivity of 8.7 μg tested up to 100 μg, which enabled visualization of proteins following SDS-PAGE. Collectively, these results demonstrate the utility of 2,2,2-trichloroethanol-based protein quantification and demonstrates the protein visualization in polyacrylamide gels based on 2,2,2-trichloroethanol-labeling pre-electrophoresis.

Protein quantification. The Bradford method was utilized with few modifications indicated 1 . To 2 μL of protein sample, 198 μL of Bradford reagent (0.1 mg/mL Coomassie Brilliant Blue G-250, 5% (v/v) methanol, 8.5% H 3 PO 4 ) was added and incubated at room temperature for 5 minutes. Absorbance was measured at 595 nm. For UV A280 experiments, the absorbance at 280 nm of 100 μL BSA standard solutions was measured in a UV-transparent microplate.
All experiments involving the reaction of TCE with proteins or amino acids followed the same workflow: samples were first incubated with TCE under a 15 W UV-lamp followed by fluorescent measurements with a BioTek Cytation 5 microplate reader. The initial characterization of the fluorescence emission of the reaction products (emission ʎ = 350-600 nm and excitation ʎ = 310 nm) and excitation spectra (emission ʎ = 450 nm and excitation ʎ = 250-400 nm) utilized 5-minute incubations of the samples with a final assay concentration of 0.5% (v/v) TCE under UV-light. To achieve this, 90 μL of TCE reagent (0.56% (v/v) TCE in PBS) was incubated with 10 μL of 0-1 μg/μL BSA, glycine, L-phenylalanine, and L-tryptophan as well as 0-0.4 μg/μL L-tyrosine.
Optimization of TCE concentration and UV-exposure time were done using BSA protein as a standard at final assay concentrations of 0.5 µg/µL and 1.0 µg/µL. TCE concentration optimization was performed with a 5-minute UV-exposure time and final assay concentrations of 0-2% (v/v) TCE, followed by fluorescence intensity readings (emission ʎ = 450 nm and excitation ʎ = 310 nm; emission ʎ = 280 nm and excitation ʎ = 350 nm). Optimization of UV-exposure time with the 15 W UV-lamp utilized a final assay concentration of 0.5% (v/v) TCE and 0-30-minute incubation while monitoring the fluorescence emission at ʎ = 450 nm with an excitation at ʎ = 310 nm.
The final TCE assay incorporated the same volumes of TCE reagent and sample described previously, resulting in a final TCE assay concentration of 0.5%, and a 15-minute UV-exposure time followed by the measurement of fluorescence emission at ʎ = 450 nm with an excitation at ʎ = 310 nm.
Low-volume TCE assay and SDS-PAGE visualization. The total volume of the TCE assay was reduced to 20 μL while maintaining the same volumes of sample and original TCE stock solution. To 10 μL of protein solution, an equal volume of TCE Ultra Reagent (5% (v/v) TCE in PBS) was added followed by 0-15 minutes of UV-exposure while monitoring the fluorescence emission at ʎ = 450 nm with an excitation at ʎ = 310 nm. Subsequently, the assay was diluted in 2 X Laemmli Sample Buffer (Bio-Rad Laboratories), heated at 95 °C for 5 minutes, and 30 μL of diluted solution (i.e. 75% of total protein) was subjected to SDS-PAGE on a 10% www.nature.com/scientificreports www.nature.com/scientificreports/ resolving gel at 120 V for 1.5 hours. The fluorescence of TCE-modified protein was visualized directly on a BioRad ChemiDoc TM XR+ System using a UV-light box. Furthermore, proteins in the same gel were visualized after incubations of the gel in Coomassie Brilliant Blue (CBB) staining solution (1 mg/mL CBB R250, 50% (v/v) methanol, 10% (v/v) glacial acetic acid) and destaining solution (50% (v/v) methanol, 10% (v/v) glacial acetic acid).

Results and Discussion
Protein fluorescent spectra. The majority of protein quantification methods exploit chemical interactions between reagent compounds and proteins, generating detectable and quantifiable signals that are proportional to concentration. The Bradford method and Lowry assay are two widely used protein quantification assays that result in colorimetric shifts, enabling quantification based on absorbance 1,2 . These assays are based on the colour  www.nature.com/scientificreports www.nature.com/scientificreports/ changes of bound vs unbound anionic dyes (i.e. CBB) and copper-based reactions, respectively. The TCE-based methodology presented herein extends the range of rapid and sensitive protein quantification methods that are currently available to researchers.
Detection and quantification of proteins in polyacrylamide gels with trichlorinated compounds relies on UV-dependent covalent modification of the tryptophan indole ring with chemical groups derived from such trichlorinated compounds 3,4 . This modification results in a red-shifted fluorescent emission extended into the visible range, allowing for visualization of protein bands with a gel imaging system. Specifically, the reaction involving TCE yields an acylated indole ring (Fig. 1) and has been used widely in visualization of protein in polyacrylamide gels 3,4 . The TCE assay described herein exploits this photochemical modification to generate a quantification curve for protein concentration as a function of TCE-reacted protein fluorescence. BSA was chosen as the analytical standard as it is commonly used for this purpose across different quantification methods and also has a typical ~3% tryptophan residue content.
Initially, the fluorescent properties of the reaction products were characterized prior to optimization of TCE percentage and UV-exposure time. For this purpose, 0.5% (v/v) TCE was used as this percentage was demonstrated to be optimal in SDS-PAGE band detection 4 . Additionally, from lab usage and previous literature 4 it is known that signals in SDS-PAGE generally appear and saturate within less than 5 minutes of UV-exposure, therefore a 5-minute reaction time was used for all subsequent experiments involving characterization of protein fluorescence. At an excitation of 310 nm, the emission monitored between 350-600 nm displayed a maximum at 450 nm, which was absent in unreacted protein (Figs 2 and S1). Furthermore, the fluorescence intensity at 450 nm increased in a dose-dependent manner with protein concentration (Fig. 2B). Natural tryptophan fluorescence at an emission of 350 nm could be observed in unreacted protein, whereas the fluorescence at this wavelength greatly diminished in the presence of 0.5% (v/v) TCE (Fig. 2). Although TCE has been demonstrated to be a quencher of protein fluorescence in the absence of intentional UV exposure 14 , the shift of peak fluorescence emission from 350 nm to 450 nm upon incubation with TCE under UV-exposure is an indicator of tryptophan labeling.
The incubation of BSA with TCE altered the excitation spectra monitored between 250-400 nm at a 450 nm fluorescence emission (Fig. 2). Within these parameters, TCE-reacted BSA displayed increasing fluorescence www.nature.com/scientificreports www.nature.com/scientificreports/ intensity with protein concentration, whereas unreacted BSA failed to be reliably detected. More specifically, the former displayed two observable peaks at 310 nm and 355 nm excitation wavelengths with comparable fluorescence intensities at 450 nm emission (Fig. 2B). Similarly, excitation peaks at 315 nm and 355 nm for TCE-reacted calf-unwinding protein (UP1) had previously been observed at a 455 nm emission 7 . Although one excitation peak was anticipated due to the number of applications in literature referring to the use of TCE as a label for only the tryptophan indole ring, we speculated that other UV-excitable residues may react with TCE to yield additional products with red-shifted fluorescent properties. Indeed, N-acetyltyrosineamide has been demonstrated to react with TCE and yield a product with altered fluorescent spectra 14 . Therefore, the two excitation peaks result from reactions of TCE with both tryptophan and tyrosine residues to yield semi-overlapping fluorescent spectra which vary between proteins due to variance in amino acid content. This may be confirmed by obtaining the fluorescent spectra of TCE-reacted proteins that lack either tryptophan or tyrosine residues.
Amino acid reactivities. The reaction of TCE with tryptophan is dependent on the indole ring entering an excited electron state, which is attainable via UV-irradiation. Other aromatic amino acids such as phenylalanine and tyrosine are also UV-excitable, therefore we explored the reactivity of TCE with aromatic amino acids through the generation of products with fluorescent emission between 350-600 nm. As two observable www.nature.com/scientificreports www.nature.com/scientificreports/ peaks were present in the excitation spectra of TCE-reacted protein at 310 nm and 355 nm (Fig. 2B), we examined the TCE-reacted amino acid emission spectra at both excitation wavelengths. At 310 nm excitation, the fluorescence emission intensity of TCE-reacted tyrosine and tryptophan increased in a dose-dependent manner with amino acid concentration (Fig. 3C,D), whereas incubation of TCE with glycine and phenylalanine under UV-light did not yield any detectable fluorescent products at measured concentration (Fig. 3A,B). Additionally, only TCE-reacted tryptophan displayed an observable dose-dependent relationship between amino acid concentration and fluorescence emission intensity at 355 nm excitation (Fig. S2). In agreement with the fluorescent emission spectra of unreacted and TCE-reacted BSA, unreacted tyrosine and tryptophan amino acids displayed little to no observable fluorescence in comparison to the corresponding TCE-reacted amino acids at peak wavelengths (Fig. S3). As 310 nm excitation light yields fluorescent signal from both TCE-reacted tryptophan and tyrosine residues, this excitation wavelength was used in subsequent experiments as it allows for quantification based on multiple amino acid residues. Assay optimization. Following characterization of UV-dependent TCE-labeling of protein and amino acids, a re-optimization of TCE concentration and UV-exposure time was performed to determine optimal reaction conditions providing maximal sensitivity at 310/450 nm emission/excitation fluorescence (i.e. TCE-reacted protein fluorescence). Initially, TCE concentration was varied between 0-2% (v/v) while fixing UV-exposure time at 5 minutes and protein concentration at 0.5 µg/µL. TCE-reacted protein fluorescence increased in a dose-dependent manner with TCE concentration to a maximal signal and plateau after 0.1% (v/v) TCE (Fig. 4A). An opposing relationship was observed for natural indole fluorescence, plateauing to a minimum after 0.5% (v/v) TCE (Fig. 4B). The decrease in natural indole fluorescence was expected as covalent modification of the tryptophan ring with trichlorinated compounds results in a shifted fluorescent emission, as was demonstrated through comparisons of unreacted and TCE-reacted emission spectra (Fig. 2). Thus, a final TCE concentration of 0.5% (v/v) was chosen for subsequent experiments as minimal natural indole fluorescence and maximal TCE-reacted protein fluorescence are ideal criteria to be satisfied. Finally, UV-exposure time was varied between 0-30 minutes, while fixing TCE concentration constant at 0.5% (v/v) and protein concentration at 0.5 µg/µL and 1.0 µg/µL. An optimal UV-exposure time of 15 minutes was chosen for subsequent experiments as maximal TCE-reacted protein fluorescence occurred at this time point (Fig. 4C). Furthermore, it could be observed that 0.5% (v/v) TCE was in excess for both 0.5 µg/µL and 1.0 µg/µL protein as the TCE-reacted protein fluorescence plateaued in the same time frame for both protein concentrations. Although further optimization for TCE concentration using a 15-minute UV-exposure time would likely demonstrate an optimal concentration lower than 0.5% (v/v), using this concentration ensures the reagent is sufficiently available in excess.
Quantification curves. The utility of this UV-dependent TCE-based protein modification was demonstrated through the construction of quantification curves displaying the relationship between protein amount and detectable signal for TCE, Bradford, and A280 assays (Fig. 5). The utility of the Bradford assay was limited as the detectable linear range was found to be restricted to 0.22-3 μg and the signal quickly saturated beyond this range (Fig. 5C). In contrast, the TCE assay had an LOD of 10.5 μg within a linear range up to 200 μg (i.e., the maximum protein amount tested), displaying an LOD 46% lower than the comparable LOD for A280 of 23 μg (Fig. 5A,B). Overall, the TCE-based protein quantification assay was found to be advantageous as a medium to high range protein quantification assay with greater sensitivity in comparison to the standard A280 assay. require the use of additional reagents, such as the A280 assay, are an attractive option for small or precious samples as they may be recovered and re-used for downstream applications. Similarly, as the TCE-modification of protein enables visualization in polyacrylamide gels, we aimed to extend the re-use of protein previously used for quantification for SDS-PAGE. To enable practical use of the TCE-modified protein for this application, we developed a low-volume TCE assay with a 20 μL final volume. The low-volume TCE assay maintained greater sensitivity over the A280 assay, displaying an LOD of 8.7 μg after 15 minutes of UV-exposure (Fig. S4). Microplate assay samples were then subsequently subjected to SDS-PAGE. After separation of the TCE-modified protein by SDS-PAGE, the modified protein was successfully resolved and visualized upon UV-illumination in comparison to an unlabeled control BSA sample (Fig. 6A). Coomassie staining of the gel, post SDS-PAGE, served as visual confirmation and control of protein loading (Fig. 6B). Thus, the low-volume TCE assay enables sample use and direct visualization in SDS-PAGE analysis. As previous reports utilize TCE incorporated into the SDS-PAGE gel matrix during gel casting, the use of this visualization method with most commercially available pre-cast gels would be restricted to procedures involving soaking of the gel in TCE solutions post-electrophoresis. In contrast, the methodology outlined in this study involving the pre-electrophoresis labeling and quantification of proteins with TCE, may present an attractive option due to the volume of TCE saved in comparison to soaking of gels in millilitre volumes of solution.

Conclusion
We have presented a new microplate format protein quantification assay based on the covalent modification of protein with TCE. The method allows researchers to quantify precious samples using an assay more sensitive than the standard A280 method while maintaining the option of sample re-use for SDS-PAGE. Finally, the potential of TCE-labeling pre-electrophoresis enables practical use of this fluorescent visualization method with both homemade and pre-cast polyacrylamide gels, which may further its use as a visualization method for applications such as two-dimensional gel spot analysis or total protein normalization prior to immunoblot transfer.